The present invention relates to the preparation of protective antigens by recombinant DNA technology for use as anthelmintic agents and as protective immunogens in the control of diseases caused by helminth parasites.
Helminth parasites are responsible for a wide range of diseases and infestations of domestic animals which, leading as they do to loss of production and even animal mortality, are of considerable economic importance. Thus for example, the blood feeding nematode Haemonchus infects the lining of the gastrointestinal tract of ruminants, causing anaemia and weight loss and if untreated frequently leads to death. Animals infected with the related non-blood feeding nematode Ostertagia similarly fail to thrive and may die if untreated. Other genera of helminths of economic importance include Trichostrongylus and Nematodirus which cause enteritis in various animals, and trematodes.
Problems are also caused by nematodes such as hookworms (eg. Necator, Ancylostoma, Uncinaria and Bunostomum spp) and flukes (eg. Fasciola, Paramphistomum and Dicrocoelium) and their relatives which in addition to ruminants and domestic pets, also infect humans, frequently with fatal results.
Control of nelminth parasites presently relies primarily on the use of anthelmintic drugs combined with pasture management. Such techniques have a number of drawbacks howeverxe2x80x94frequent administration of drugs and pasture management are often not practical, and drug-resistant helminth strains are becoming increasingly widespread.
There is therefore a need in this field for an effective anti-helminth vaccine and many efforts have been concentrated in this area in recent years. However, as yet there are no commercially available molecular or sub-unit vaccines for the major helminth species, particularly for the gastrointestinal nematodes of ruminants, such as Haemonchus and Ostertagia.
Most promising results to data have been obtained with novel proteins isolated from Haemonchus, which have potential as protective antigens not only against Haemonchus but also against a range of other helminths. In particular the protein doublet H11OD, found at the luminal surface of the intestine of H.contortus has been shown to confer protective immunity against haemonchosis in sheep.
H11OD from H.contortus has an approximate molecular weight of 110 kilodaltons (kd) under reducing and non-reducing conditions, as defined by SDS-PAGE, and is described in WO88/00835 and WO90/11086. The term xe2x80x9cH11ODxe2x80x9d as used herein refers to the protein doublet H11OD as defined in WO88/00835 and WO90/11086. Corresponding proteins have also recently been shown in other helminth species, eg. Necator americanus. 
A number of methods for the purification of H11OD have been described in WO88/00835 which suffice for the characterisation of the protein, and may be scaled up to permit production of the protein in experimentally and commercially useful quantities. There is however a need for an improved and convenient source from which to prepare not only H11OD but also related antigenic proteins, especially for a process based on recombinant DNA technology and expression of the proteins in suitably transformed prokaryotic or eukaryotic organisms.
The present invention seeks to provide such an improved procedure. Sequence determination of cDNAs for H11OD from Haemonchus contortus has been performed and the predicted amino acid sequences have been found to display homology with a family of integral membrane aminopeptidases (systematic name: xcex1-amino acyl peptide hydrolase (microsomal)).
The mammalian integral membrane aminopeptidases are located in several tissues, eg. on the microvillar brush border of intestines, and kidney. Their role in the kidney is unclear, but in the intestine their functional is to cleave the small peptides which are the final products of digestion (for reviews, see Kenny and Maroux, 1982; Kenny and Turner, 1987; Noren et al, 1986; Semenza, 1986).
In one aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences which encode helminth aminopeptidase enzymes or antigenic portions thereof substantially corresponding to all or a portion of the nucleotide sequences as shown in FIGS. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or sequences coding for helminth aminopeptidase enzymes which are substantially homologous with or which hybridise with any of said sequences.
A nucleic acid according to the invention may thus be a single or double stranded DNA, cDNA and RNA.
Variations in the aminopeptidase-encoding nucleotide sequences may occur between different strains of helminth within a species, between different stages of a helminth life cycle (e.g. between larval and adult stages), between similar strains of different geographical origin, and also within the same helminth. Such variations are included within the scope of this invention.
xe2x80x9cSubstantially homologousxe2x80x9d as used herein includes those sequences having a sequence identity of approximately 50% or more, eg. 60% or more, and also functionally-equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion. By xe2x80x9cfunctionally equivalentxe2x80x9d is meant nucleic acid sequences which encode polypeptides having aminopeptidase activities which are similarly immunoreactive ie. which raise host protective antibodies against helminths.
Nucleic acid molecules which hybridise with the sequences shown in FIGS. 2, 3, 4 or 5 (composed of SEQ ID NOS: 1 to 15) or any substantially homologous or functionally equivalent sequences as defined above are also included within the scope of the invention. xe2x80x9cHybridisationxe2x80x9d as used herein defines those sequences binding under non-stringent conditions (6xc3x97SSC/50% formamide at room temperature) and washed under conditions of low stringency (2xc3x97SSC, room temperature, more preferably 2xc3x97SCC, 42xc2x0 C.) or conditions of higher stringency eg. 2xc3x97SSC, 65xc2x0 C. (where SSC=0.15M NaCl, 0.015M sodium citrate, pH 7.2).
Methods for producing such derivative related sequences, for example by site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids are well known in the art, as are methods for determining whether the thus-modified nucleic acid has significant homology to the subject sequence, for example by hybridisation.
Provision of a nucleic acid molecule according to the invention thus enables recombinant aminopeptidase enzymes, or immunogenic fragments thereof, to be obtained in quantities heretofore unavailable, thereby permitting the development of anti-helminth vaccines.
In another aspect the present invention thus provides nucleic acid molecules comprising one or more nucleotide sequences encoding one or more polypeptides capable of raising protective antibodies against helminth parasites, which sequences incorporate one or more antigenic determinant-encoding regions from the aminopeptidase-encoding sequences as shown in FIGS. 2, 3, 4 or 5 (composed from SEQ ID NOS: 1 to 15).
The present invention also extends to synthetic polypeptides comprising one or more amino acid sequences constituting an aminopeptidase enzyme or antigenic portions thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15), or a functionally-equivalent variant thereof other than a synthetic polypeptide corresponding to the protein doublet H11OD, or a synthetic polypeptide corresponding to any of the individual polypeptide sequences disclosed in WO90/11086.
Alternatively viewed, the invention also provides synthetic polypeptides comprising an amino acid sequence constituting an aminopeptidase enzyme or an antigenic portion thereof, substantially corresponding to all or a portion of the nucleotide sequences as shown in FIG. 2, 3, 4 or 5 (SEQ ID NOS: 1 to 15) or a functionally-equivalent variant thereof, substantially free from other Haemonchus contortus components.
The invention further extends to vaccine compositions for stimulating immune responses against helminth parasites in a human or non-human animal, comprising at least one synthetic polypeptide as defined above, together with a pharmaceutically acceptable carrier.
WO90/11086 discloses a number of polypeptide or partial polypeptide sequences (SEQ ID NOS: 25-54) obtained by proteolytic digestion or chemical cleavage of the protein doublet H11OD as follows:
Uncertainties are shown either by the form Phe/Gly, where the first three letter codes represents the most likely correct amino acid based on the strength of the signal, or by a question mark; a sign xe2x80x9cxe2x88x92xe2x80x9d means an unknown residue.
The specific individual polypeptide sequences which are disclosed in WO09/11086 are disclaimed.
The term xe2x80x9cpolypeptidexe2x80x9d as used herein includes both full length protein, and shorter peptide sequences.
xe2x80x9cFunctionally equivalentxe2x80x9d as used above in relation to the polypeptide amino acid sequences defines polypeptides related to or derived from the above-mentioned polypeptide sequences where the amino acid sequence has been modified by single or multiple amino acid substitution, addition or deletion, and also sequences where the amino acids have been chemically modified, including by glycosylation or deglycosylation, but which nonetheless retain protective antigenic (immunogenic) activity. Such functionally-equivalent variants may occur as natural biological variations or may be prepared using known techniques, for example functionally equivalent recombinant polypeptides may be prepared using the known techniques of site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of amino acids.
Generally, the synthetic polypeptides according to the invention represent protective antigenic sequences. The term xe2x80x9cprotective antigenxe2x80x9d as used herein defines those antigens capable of generating a host-protective (immunogenic) immune response ie. a response by the host which leads to the generation of immune effector molecules, antibodies or cells which sterilise the fecundity of, damage, inhibit or kill the parasite and thereby xe2x80x9cprotectxe2x80x9d the host from clinical or sub-clinical disease and loss of productivity. Such a protective immune response may commonly be manifested by the generation of antibodies which are able to inhibit the metabolic function of the parasite, leading to stunting, lack of egg production and/or death.
The synthetic polypeptides according to this aspect of the invention may be prepared by expression in a host cell containing a recombinant DNA molecule which comprises a nucleotide sequence as broadly described above operatively linked to an expression control sequence, or a recombinant DNA cloning vehicle or vector containing such a recombinant DNA molecule. Alternatively the polypeptides may be expressed by direct injection of a naked DNA molecule according to the invention into a host cell.
The synthetic polypeptide so expressed may be a fusion polypeptide comprising a portion displaying the immunogenicity of all or a portion of an aminopeptidase enzyme and an additional polypeptide coded for by the DNA of the recombinant molecule fused thereto. For example, it may be desirable to produce a fusion protein comprising a synthetic aminopeptidase or other polypeptide according to the invention coupled to a protein such as xcex2-galactosidase, phosphatase, glutathione-S-transferase, urease, hepatitis B core antigen (Francis et al., 1989) and the like. Most fusion proteins are formed by expression of a recombinant gene in which two coding sequences have been joined together with reading frames in phase. Alternatively, polypeptides can be linked in vitro by chemical means. All such fusion or hybrid derivatives of aminopeptidase-encoding nucleic acid molecules and their respective amino acid sequences are encompassed by the present invention. Such suitable recombinant DNA and polypeptide expression techniques are described for example in Sambrook et al., 1989. Alternatively, the synthetic polypeptides may be produced by chemical means, such as the well-known Merrifield solid phase synthesis procedure.
Further aspects of the invention include use of a nucleic acid molecule or a synthetic peptide or polypeptide as defined above, for the preparation of a vaccine composition for stimulating immune responses in a human or non-human, preferably mammalian animal against helminth parasite infections.
Alternatively viewed, the invention also provides a method of stimulating an immune response in a human or non-human, preferably mammalian, animal against a helminth parasite infection comprising administering to said animal a vaccine composition comprising one or more polypeptides encoded by a nucleotide sequence as defined above.
A vaccine composition may be prepared according to the invention by methods well known in the art of vaccine manufacture. Traditional vaccine formulations may comprise one or more synthetic polypeptides according to the invention together, where appropriate, with one or more suitable adjuvants eg. aluminium hydroxide, saponin, QuilA, or more purified forms thereof, muramyl dipeptide, mineral oils, or Novasomes, in the presence of one or more pharmaceutically acceptable carriers or diluents. Suitable carriers include liquid media such as saline solution appropriate for use as vehicles to introduce the peptides or polypeptides into a patient. Additional components such as preservatives may be included.
An alternative vaccine formulation may comprise a virus or host cell eg. a microorganism (eg. vaccinia virus, adenovirus, Salmonella) having inserted therein a nucleic acid molecule (eg. a DNA molecule) according to this invention for stimulation of an immune response directed against polypeptides encoded by the inserted nucleic acid molecule.
Administration of the vaccine composition may take place by any of the conventional routes, eg. orally or parenterally such as by intramuscular injection, optionally at intervals eg. two injections at a 7-28 day interval.
As mentioned above, the amino acid translation of the nucleotide sequences depicted in FIGS. 2, 3, 4 or 5 show sequence homology with a family of integral membrane aminopeptidase enzymes. This was determined by searching various databases available in the Genetics Computer Group Sequence analysis software package, version 7.01, November 1991 (Devereux et al., (1984)), using translations of the sequences shown in FIGS. 2, 3, 4 or 5. Two such comparisons are shown in FIG. 6.
Expression of the aminopeptidase-encoding sequences according to the invention can, as mentioned above, be achieved using a range of known techniques and expression systems, including expression in prokaryotic cells such as E.coli and in eukaryotic cells such as yeasts or the baculovirus-insect cell system or transformed mammalian cells and in transgenic animals and plants. Particularly advantageously, the nucleotide sequences may be expressed using the transgenic nematode system such as the system for the nematode Caenorhabditis described for example in Fire, (1986); Fire et al., (1989); Spieth et al., (1988); Han et al., (1990).
A further aspect of the invention provides a method for preparing a synthetic polypeptide as defined above, which comprises culturing a eukaryotic or prokaryotic cell containing a nucleic acid molecule as defined above, under conditions whereby said polypeptide is expressed, and recovering said polypeptide thus produced.
Further aspects of the invention thus include cloning and expression vectors containing nucleotide sequences according to the invention. Such expression vectors include appropriate control sequences such as for example translational (eg. start and stop codes) and transcriptional control elements (eg. promoter-operator regions, ribosomal binding sites, termination stop sequences) linked in matching reading frame with the nucleic acid molecules of the invention.
Vectors according to the invention may include plasmids and viruses (including both bacteriophage and eukaryotic viruses) according to techniques well known and documented in the art, and may be expressed in a variety of different expression systems, also well known and documented in the art. Suitable viral vectors include, as mentioned above, baculovirus and also adenovirus and vaccinia viruses. Many other viral vectors are described in the art.
A variety of techniques are known and may be used to introduce such vectors into prokaryotic or eukaryotic cells for expression, or into germ line or somatic cells to form transgenic animals. Suitable transformation or transfection techniques are well described in the literature.
Transformed or transfected eukaryotic or prokaryotic host cells or transgenic organisms containing a nucleic acid molecule according to the invention as defined above, form a further aspect of the invention.
Uekaryotic systems in general, and the nematode expression system in particular, have the advantage that post-translational processing, and particularly glycosylation can occurxe2x80x94in the case of the transgenic nematode system, a glycosylation corresponding to that found in the native protein may be expected. This represents an important aspect of the invention, since in many cases post-translational processing is required for the recombinant protein to express optimum biological activity.
Mammalian cell expression systems, also have a number of advantages. Mammalian host cells provide good reproduction of the native form and protective epitopes of the antigen since a eukaryotic expression system will give rise to more similar glycosylation patterns, disulphide bonding and other post-translational modifications than E.coli which may produce an insoluble protein requiring refolding and having poor reproduction of the native form. In addition mammalian glycosylation is unlikely to induce an immune response which distracts from a protective anti-protein response. For protection of humans and domestic animals, it is thus preferable to use human or animal fibroblast or myeloma cell lines such as HeLaxe2x80x94a human cell line; GHKxe2x80x94baby hamster kidney cells; VERO, a monkey kidney cell line; FR3T3, Fisher rat fibroblasts; NIH3T3, a mouse fibroblast cell line; C127I, a mouse mammary tumour cell line; CV-1, African green monkey kidney fibroblasts; 3T6, mouse embryo fibroblasts; L cells, a mouse cell line; CHO, a Chinese Hamster Ovary cell line; NSO NSI, SP2 and other mouse myeloma cell lines and rat myeloma cell lines such as YB2/0 and Y3.
Vectors appropriate for different classes of mammalian cell lines are well known in the art. In general, these will comprise a promoter and/or enhancer operably connected to a nucleotide sequence encoding the antigen or fragment thereof. Suitable promoters include SV40 early or late promoter, eg. PSVL vector, cytomegalovirus (CMV) promoter, mouse metallothionein I promoter and mouse mammary tumour virus long terminal repeat. The vector preferably includes a suitable marker such as a gene for dihydrofolate reductase or glutamine synthetase. Vectors of those types are described in WO86/05807, WO87/04462, WO89/01036 and WO89/10404.
Transfection of the host cells may be effected using standard techniques, for example using calcium phosphate, DEAE dextran, polybrene, protoplast fusion, liposomes, direct microinjection, gene cannot or electroporation. The latter technique is preferred and methods of transfection of mammalian cell lines using electroporation are described by Andreason et al., 1980. In general, linear DNA is introduced more readily than circular DNA.
In the case of the protein H11OD, it has been found to have a unique and unusual glycosylation pattern, which is thought to contribute to immunoactivity since many monoclonal antibodies so far obtained to H110D from Haemonchus recognise carbohydrate epitopes which may be of importance in developing useful vaccines.
In particular the following glycosylation pattern for H110D from Haemonchus has been demonstrated:
i. about 65% of oligosaccharides are N-linked, the remainder O-linked;
ii. the major part (eg. about 48%) of the N-linked oligosaccharide is of the complex class;
iii. substantially all (eg. greater than 95%) of the oligosaccharides are uncharged;
iv. the relative molar content of the constituent monosaccharides is N-acetylgalactosamine 1.0, fucose 3.6, galactose 4.1, glucose 4.4, mannose 6.2 and N-acetylglucosamine 5.2;
v. the oligosaccharides, other than the major oligosaccharide (designated oligosaccharide D), are substantially resistant to degradation by a broad range of exo-glycosidase (eg. xcex1-D-mannosidase, xcex2-D-mannosidase, xcex2-D-glucosidase, xcex2-D-galactosidase, xcex1-D-galactosidase, xcex1-L-fucosidase, xcex2-D-xylosidase, xcex2-D-acetylglucosaminidase).
Such oligosaccharides and glycosproteins containing them form a further aspect of this invention.
Oligosaccharide D of the Haemonchus H110D glycoprotein is of the N-linked type and has a novel structure consisting of two fucose residues attached by an xcex1-1,3 linkage and an xcex1-1,2 linkage to a mannose (N-acetylglucosamine)2 core.
Another aspect of the invention thus provides an oligosaccharide having the structure: 
and more particularly the structure: 
especially when linked to a protein, eg. a recombinant protein such as a helminth aminopeptidase protein or an antigenic fragment thereof, or when used to generate anti-idiotypic antigens for immunisation especially of very young animals.
Animal glycoproteins generally have fucose xcex1-1,6 linkages and the fucose xcex1-1,3 linkage of the oligosaccharide of the present invention is an unusual feature.
This invention will now be described in more detail with particular reference to the protein H110D from Haemonchus contortus. However, by a variety of techniques such as histochemistry and DNA hybridisation, H110D equivalents have been observed in other parasite species. It is believed that the H110D protein is a multigene complex and that in addition, the nucleotide sequences encoding it, may exhibit sequence variations between different strains and different life cycle stages of the helminth. Moreover there may exist multiple enzyme forms (isoenzymes) which may be differentially expressed at different stages, or in different strains. In this study DNA sequences, and thus the predicted amino acid sequences, have been determined from cDNA clones and PCR products obtained from mRNA corresponding to the H110D gene by recombinant DNA technology from different sources, and at different parasitic stages of H. contortus life cycle.
Sequencing of cDNA and PCR products has enabled us to identify three closely related H110D sequences which are here designated H11-1 (SEQ ID NO: 19), H11-2 (SEQ ID NO: 20) and H11-3 (SEQ ID NO: 21). H11-1 comprises three contiguous and overlapping sequences, cDNA clone AustB1 (SEQ ID NO: 6), PCR product A-648 (SEQ ID NO: 9) and at the 3xe2x80x2 end PCR product 014-178 (SEQ ID NO: 12); H11-2 comprises the PCR products A-650 and 2.5 kb (SEQ ID NOS: 10 and 7 respectively); H11-3 comprises the PCR products 3.5 kb and A-649 (SEQ ID NOS: 8 and 11 respectively). The specific relationships between the individual sequenced cDNA and PCR product clones and H11-1, -2 and -3 are summarised in FIG. 1 and shown in detail in FIGS. 3, 4 and 5.
Differences and variations in the sequences obtained from the cDNA clones and PCR products have been observed, as can be seen in particular from FIGS. 2, 3, 4 and 5 (composed of SEQ ID NOS: 1 to 15 and 19 to 21) and as summarised in Table 1.
The differences can be attributed to different mRNAs (of the multigene family). In addition, the variations may be due, at least in part, to different variants of the H110D-encoding sequence or mRNA present at different stages of the lift cycle or in strains differing in geographical origin.
Table 2 additionally shows levels of identity and similarity between the corresponding predicted amino acid sequences and two published mammalian aminopeptidase sequences.